Memno peptides, process for their preparation and use thereof

ABSTRACT

The invention relates to novel peptide derivatives, called memno peptides, of the formula (I):                    
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and (A)n have the meaning herein, obtainable by cultivation of  Memnoniella echinata  FH2272, DSM 13195 or mutants and variants of this, a process for their preparation and the use of the compounds as pharmaceuticals, for example against cardiac insufficiency.

The invention relates to novel peptide derivatives, called memnopeptides, obtainable by fermentation of Memnoniella echinata FH2272, DSM13195, in a culture medium, a process for the preparation of the memnopeptides, and the use of the memno peptides as pharmaceuticals, forexample, for the production of a pharmaceutical for the treatment ofcardiac insufficiency.

Cardiovascular disorders still rank first as causes of death in thewestern industrial countries. A not inconsiderable proportion of theseare patients with the diagnosis of cardiac insufficiency. Cardiacinsufficiency is understood as meaning the inadequate functioning of theheart. The heart is not able to produce an output corresponding to themammal's requirements. Cardiac insufficiency is an acute or chronicinability of the heart, under load or even at rest, to muster the bloodoutput necessary for metabolism or to take up the venous return. It isthe state of the heart wherein the compensation mechanisms, such asheart rate, stroke volume, or hypertension, no longer suffice for themaintenance of a normal cardiac output. Cardiac insufficiency has avariety of causes, for example inflammatory and degenerative myocardial(heart muscle) changes, coronary circulatory disorders and cardiacinfarct. Cardiac insufficiency leads to changes in the peripheralcirculation, to impairment of the respiration, the kidney function, andthe electrolyte balance, and also to reduced power of the skeletalmusculature, and in the end it frequently leads to death.

Cardiac insufficiency generally occurs at an advanced age. The incidenceis 3 disorders per 1000 inhabitants per year in 35-64 year-olds and10/1000/year in the age group from 65 to 94 years. Mortality increasesin 75-year-olds almost by a factor of 200 compared with the age groupbetween 35 and 44 years. The mortality rate has remained approximatelyconstant between 1970 and 1983, as investigations in the USA showed. Forthe Federal Republic of Germany, the same numbers are to be assumed.More than 50% of patients die in the first five years after diagnosis.This statistical examination, in and of itself, shows the greatimportance of cardiac insufficiency for the population, but it alsoconfirms the inadequate possibilities of medicinal treatment which areavailable to the physician today.

In view of the inadequacy of present treatments, new concepts have beendeveloped which should lead to innovative cardiac remedies. The abilityof the cardiac and skeletal muscles to contract and thus to performmechanical work is dependent on (1) contractile structural elements(myofibrils) and (2) chemical energy (ATP) available to the myofibrils,which is converted into mechanical energy in the contraction process.Shortening of the myofibrils occurs in the contraction process. This maybe initiated by motor nerve impulses, under the action of which calciumions (Ca²⁺) enter into the sarcoplasmatic space from the extracellularspace within a few milliseconds and the calcium depots are emptied. Inmyocardial insufficiency (cardiac insufficiency), the Ca²⁺ concentrationin myofibrils may be reduced. Ca²⁺ ions, however, are indispensable forthe activation of the contractile apparatus. If there is increaseddemand, Ca²⁺ is generally pumped into the sarcoplasmatic reticulum (SR)under catalysis of a membranous Ca²⁺-dependent Mg²⁺-ATPase: this enzymeis also called Sarco(Endo)plasmatic Reticulum Ca²⁺ATPase (SERCA2).According to hydropathic analysis, the Ca²⁺ATPase comprises tentransmembranous helices and a number of extramembranous loops. On thecytosolic side, domains for Ca²⁺ and ATP binding, for phosphorylationand for interaction with the modulator protein phospholamban (PLB) areformed. The latter is a protein pentamer, which is localized in themembrane of the SR and exerts an inhibitory influence on SERCA2 in theunphosphorylated state. Under physiological stress, a phosphorylation ofPLB takes place, which increases the Ca²⁺ affinity of SERCA2a and thusincreases the transport rate for Ca²⁺ ions in the SR. Thephosphorylation of PLB (a 52 amino acid protein) takes place on twoamino acid residues: serine-16 may be phosphorylated by thecAMP-dependent protein kinase and threonine may be phosphorylated inposition 17 by the Ca²⁺/calmodulin-dependent kinase. Thisphosphorylation causes a change in confirmation in PLB followed by anincreased affinity of SERCA2 for Ca²⁺. Anti-PLB antibodies are able toimitate the PLB phosphorylation effect and thus confirm the key role ofPLB as a regulator of the contractile activity of the heart(Phospholamban: Protein Structure, Mechanism of Action and Role inCardiac Function. H. K. Simmerman and L. R. Jones, PhysiologicalReviews; Vol. 78, No. 4, 921ff, 1998). Activators of SERCA2 should thusbring about a favorable influence in cardiac insufficiency.

It has surprisingly been found that cultures of the fungal strainMemnoniellla echinata FH 2272, DSM 13195 contain natural substanceswhich are able to display favorable effects on the heart and thecirculation. The isolated active compounds, the memno peptides, arenatural substances comprising specific constituent groups. Theseconsitiuent groups include terpene units, a so-called polyketide moietyand a nitrogen-containing group.

Terpenes are naturally occurring compounds which can be interpretedformally as polymerization products of the hydrocarbon isoprene.According to the number of isoprene groups, monoterpenes (C₁₀),sesquiterpenes (C₁₅), diterpenes (C₂₀) etc. can be differentiated. Alarge number of compounds can be formed from the parent structures bysubstitution, cyclization, rearrangement, oxidation etc.; accordingly,many thousands of terpenes have been described in the literature.Nitrogen-containing compounds originating from the terpenes have alsobeen reported, but these are counted among the alkaloids (e.g. theGentiana alkaloids) [Römpp Chemie Lexikon [Römpp's ChemicalEncyclopedia], 9th Edition, Volume 6, page 4508 ff., Georg ThiemeVerlag, Stuttgart/New York, 1992]. These terpenes, however, differfundamentally from the memno peptides according to the invention,wherein terpenes do not contain a polyketide moiety with which they canbind nitrogen.

Examples of further, known nitrogen-containing terpenes are:

Stachybocins [J. Antibiotics, 48: 1396 (1995)];

Stachybotrins [Y. Nozawa et al. J. Antibiotics, 50: 635-645 (1997)];

Spirodihydrobenzofuran lactams[J. Antibiotics, 49: 13 (1996)];

Nakijiquinones [Tetrahedron, 51: 10867-10874 (1995)];

F1839-A to J are nitrogen-containing terpenes having polyketide moieties[Japanese Patent 061864133 and 08283118]. They are cholesterol esteraseinhibitors.

These terpene derivatives were synthesized from various strains of thegenera Memnoniella echinata and Stachybotrys and others. They weredescribed as antagonists of the endothelin receptor, as inhibitors ofHIV-1 protease and of cholesterol esterase and as hair tonics. Theinositol monophosphatase inhibitor L-671,776 was moreover isolated fromcultures of the strain Memnoniella echinata, ATCC 20928 [Y. K. T. Lam etal. J. Antibiotics, 45, pp.1397-1402, (1992)].

The memno peptides according to the invention have a differing spectrumof activity. A conspicuous feature is their activating effect on SERCA2and thus on the insufficient heart.

The present invention thus relates to compounds of formula (I)

wherein

R₁ and R₂ together are double bonded O, or H₂, or H and OH, or H andO—C₁—C₄-alkyl;

R₃ and R₄ together are double bonded O, or H₂, or H and OH, or H andO—C₁—C₄-alkyl;

R₈ is chosen from H, OH, C₁—C₄-alkyl and O—C₁—C₄-alkyl, such asO-methyl; R₆ is a group of formula (II)

wherein R₉ is H or a glycosidically bonded sugar, or

a group of the formula (III)

wherein R₁₀ is H or a glycosidically bonded sugar,

and wherein,

if R₆ is a group of the formula (II), then R₅ is a bond to the carbonatom C9 of the formula (II) and R₇ is H, or R₇ is a bond to the carbonatom C9 of the formula (II) and

R₅ is H, and

if R₆ is a group of the formula III, then R₅ and R₇ are H;

A is an amino acid,

n is an integer chosen from 1 to 12, wherein each A is the same ordifferent from every other A,

wherein the nitrogen atom in the isoindole ring of formula (I) is theN-terminal amine nitrogen of the first amino acid of the (A)n group;

or a salt or derivative thereof;

with the proviso that

A is an amino acid other than Glu when n is 1, and R₁ and R₂ togetherare double bonded O, and R₃ and R₄ together are H₂, and R₆ is a group offormula (II), and R₅ is a bond to the carbon atom C9 of the group offormula (II), and R₇ is H, and R₈ is H and R₉ is H.

In formula (I), (A)n can be at least one natural amino acid selectedfrom: Gly, Ala, Val, Leu, Ile, Pro, Ser, Thr, Phe, Tyr, Trp, Lys, Arg,Asp, His, Glu, Asn, Gln, Cys and Met. (A)n can be a peptide chain havingfrom 2 to 12 amino acids. In one embodiment (A)n comprises 10 aminoacids.

C₁-C₄-Alkyl is a straight-chain or branched alkyl having 1 to 4 carbonatoms, such as methyl, ethyl, i-propyl and tert-butyl.

The sugar can be a hexose, for example an aldohexose, such as mannose,glucose or galactose, which may be optionally substituted withadditional groups, such as C₁ to C₄-Alkyl or NH₂.

The present invention furthermore relates to all obvious derivatives ofthe compounds of the formula I. Derivatives are salts, reductionproducts, esters, ethers, acetals as well as amides and N-alkylationproducts, moreover all optical antipodes, diastereomers and allstereomeric forms.

In one embodiment, the compound of formula (I) has formula (IV)

where the groups R₁, R₂, R₃, R₄, R₉ and (A)n have the meaning asindicated above.

In the formula (IV), R₁ and R₂ taken together can be double bonded O, R₃and R₄ together can be H₂ and R₉ can be H.

In one embodiment, the inventive compound is memno peptide A,C₇₆H₁₀₈N₁₆O₁₈S, of the formula (IVa)

In this formula, the decalin structure having the 4-methyl and amethylene group is the terpene moiety; the substituted isoindole ring isthe ketide moiety. The amino acid sequence:

Met His Gln Pro His Gln Pro Leu Pro Pro (SEQ ID NO:1) is a part of thecasein sequence. The methionine (Met) can be oxidized to the sulfoxide.

Formula (IVb) shows a preferred spatial form:

wherein (A)n has the meaning as indicated above.

The physicochemical and spectroscopic properties of the preferredcompound according to the invention can be summarized as follows:

Memno peptide A

Appearance: colorless substance soluble in polar organic solvents and inwater. It is stable in neutral, mildly acidic medium.

Empirical formula: C₇₆H₁₀₈N₁₆O₁₈S,

Molecular weight: 1565.87 Da,

UV absorption (λ_(max)): 270 nm,

NMR data: see Table 1

The numbering of the carbon atoms and the associated NMR chemical shiftsare classified according to the numbering procedure for cyclicsesquiterpenes.

FIG. 1: Numbering of a cyclic sesquiterpene ketolide.

TABLE 1 NMR data (chemical shifts) of memno peptide A in DMSO - d₆ at310K. δ-¹³C m δ-¹H m ^(n)J_(HH) ^(n)J_(CH) (10 Hz) assignment  1 15.42 q0.668 d 1.802 1.802 Terpene-8-Me  2 15.72 q 0.978 s — 2.035, 1.763Terpene-10-Me  3 20.37 t 1.409 m 2.035, 1.538 2.035 Terpene-6 1.4602.035,  4 21.43 q 0.873 d 1.678 1.678, 1.428, 0.890 Leu⁸-δ  5 22.25 q0.827 s 0.918 0.918, 2.035 Terpene-4-Me  6 22.64 t 2.182 ddt 4.892,2.788 2.632 4.892, 2.788, 2.632 Met¹-β 2.307 ddt 22.80 t 2.19 ddt 4.869,2.757, 2.635 4.869, 2.757, 2.635 2.31 ddt  7 23.08 q 0.890 d 1.6781.678, 1.428, 0.873 Leu⁸-δ′  8 23.88 d 1.678 m 0.87, 0.88 Leu^(B)-γ br 9 23.90 t 1.764 m 0.962, 1.416, 1.871 0.978, 1.416, 3.227 Terpene-10.962 m 10 24.22 t 1.910 m 2.144, 4.588, 3.467 2.144, 1.771, 4.588,Pro⁹-γ 3.467, 3.689 11 24.32 t 1.863 m 2.031, 1.798, 3.625 4.356, 3.625,2.153, 1.792 Pro⁴-γ 12 24.37 t 1.927 m (2.153), (1.814), 3.624 4.388,3.624, 1.814 Pro⁷-γ 13 24.43 t 1.926 m 2.144, 1.840, 3.655, 4.224,3.655, 3.538 Pro¹⁰-γ 3.538 14 24.77 t 1.840 m 1.416, 1.740, 0.962 —Terpene-2 1.416 m 1.840, 0.926 15 26.51 t 1.940 m 1.719, 4.484, 2.1982.198 Gln³-β 1.719 m 1.719, 4.484, 2.198 16 26.85 t 1.927 m 1.722,4.468, 2.166 2.166 Gln⁶-β 1.722 m 1.927, 4.468, 2.166 17 27.04 t 2.976dd 4.598 — His⁵-β br 3.067 dd 18 27.09 t 2.937 dd 4.570 (4.570) His²-β3.067 dd 19 27.50 t 2.144 m 1.771, 4.578, 1.905, 1.945, 3.689, 3.467,4.578 Pro⁹-β 1.945 1.771 m 2.144, 4.578, 1.905, 1.945 20 28.34 t 2.144 m1.840, 4.223, 1.926 4.223, 3.669, 3.533 Pro¹⁰-β 1.840 m 2.144, 4.223,1.926 21 28.50 q 0.918 s 0.827 0.827 Terpene-4-Me 22 28.86 t 1.798 m4.356, 2.031, (1.863) Pro⁴-β 2.031 m 23 28.89 t 1.814 m 2.012, 4.388,(1.927) 4.388, 3.646 Pro⁷-β 1.814, 4.388, (1.927) 2.012 m 24 30.63 t1.538 m 1.430, 1.460, (1.409), 0.667 Terpene-7 m (1.802) 1.430 1.538,1.460, 1.802 25 30.64 t 2.166 t 1.927, 1.722 1.927, 1.722, 4.466, 6.807Gln⁶-γ 26 30.65 t 2.192 t 1.940, 1.719 1.940, 1.719, 4.489, 6.789 Gln³-γ27 31.62 t 3.154 d 2.792 6.598 Terpene-11 2.792 d 3.154 28 36.43 d 1.802ddq 1.538, 1.430, 0.667 0.667, 2.790, 3.156 Terpene-8 29 37.22 s — — —0.903, 0.824 Terpene-4 30 37.73 q 2.548 s — 2.682, 2.758 Met¹-ε 37.842.546 2.633, 2.783 31 39.45 d 2.035 dd 1.409, 1.460 0.903, 0.824, 0.971Terpene-5 32 40.07 t 1.428 dt 4.536, 1.678 0.890, 0.873, 4.536 Leu⁸-β40.03 33 41.71 s — — — 3.156, 2.790, 0.977, Terpene-10 (1.764), 2.035 3444.25 t 4.332 — — 4.881 Terpene-8′ 35 46.10 t 3.669 1.934 4.223, 2.147,1.934, 1.851 Pro¹⁰-δ 3.533 36 46.53 t 3.689 m 1.905, 1.936 4.588, 2.165,1.781, 1.936 Pro⁹-δ 3.467 m 37 46.81 t 3.624 m 1.927 1.814, 4.388 Pro⁷-δ38 46.87 t 3.644 m 1.863 — Pro⁴-δ 39 48.56 d 4.534 dt 7.918, 1.4287.918, 1.428, 1.678 Leu⁸-α 40 49.44 t 2.682, ddt 2.758, 2.162, 2.2894.892, 2.550 Met¹-γ 2.758 ddt 2.682, 2.162, 2.289 49.70 t 2.633, ddt2.783, 2.289, 2.162 4.869, 2.545 2.783 ddt 2.633, 2.289, 2.162 41 50.18d 4.466 dt 8.110, 1.927, 1.722 2.166 Gln⁶-α 42 50.31 d 4.484 dt 8.206,1.935, 1.707 2.182 Gln³-α 43 51.37 d 4.570 ddd 8.135, 2.975, 3.0822.975, 3.082 His⁵-α 44 51.68 d 4.590 ddd 8.496, 2.934, 3.067 2.934,3.067 His²-α 4.585 8.506, 2.934, 3.067 45 53.30 d 4.892 2.162, 2.2842.162 Met¹-α 53.62 4.869 2.162, 2.284 46 57.33 d 4.588 dd 2.144, 1.7712.144, 1.771, 1.905, Pro⁹-α 1.945, 3.684 47 58.31 d 4.223 dd 2.144,1.840 3.669, 3.553, 1.926, Pro¹⁰-α 2.144, (1.840) 48 59.20 d 4.388 dd2.012, 1.814 2.012 Pro⁷-α 49 59.44 d 4.356 dd 2.031, 1.798 3.635, 1.880Pro⁴-α 50 73.50 d 3.227 dd 1.840, 1.416 0.903, 0.824 Terpene-3 73.51 5197.93 s — — — 3.154, 2.792, 0.972, 0.668 Terpene-9 97.91 52 100.90 d6.598 s — — Terpene-3′ 100.88 6.596 53 112.62 s — — — 6.593, 4.330Terpene-5′ 112.55 54 116.92 s — — — 3.154, 2.792, 6.597 Terpene-1′116.90 55 116.95 d 7.230 s 8.655 (3.067), 2.937, 8.665 His²-ε 56 117.26d 7.322 s 8.771 3.092, 3.000, 8.771 His⁵-ε 57 129.32 s — — — 8.783,7.322, 3.083 2.976, His⁵-γ 4.585 58 129.63 s — — — 7.230, 3.064, 2.937,4.592 His⁵-γ 59 133.00 s — — — 4.330 1-4′ 132.98 60 133.63 d 8.655 s7.230 7.230 His²-δ 61 133.68 d 8.771 s 7.320 7.320 His⁵-δ 62 153.60 s —— — 6.597, 3.157, 2.792 Terpene-2′ 153.62 63 155.73 s — — — (6.597),3.154, 2.792, Terpene-6′ 4.330 64 168.11 s — — — 6.597, 4.330, 4.881Terpene-7′ 168.07 65 169.57 s — — — 4.588, (4.223) Pro⁹-CO 66 169.59 s —— — 8.110, (4.570) His⁵-CO 67 169.69 s — — — 4.534, 1.444 Leu⁸-CO 68169.74 s — — — 8.207, 2.937, 4.585 His²-CO 69 169.97 s — — — 8.513,4.869, 2.190 Met¹-CO 169.89 8.499, 4.892, 2.182 70 170.07 s — — — 4.471,1.733, 1.924, 4.356 Gln³-CO 71 170.21 s — — — 4.466, 1.722, 1.927, 4.388Gln⁶-CO 72 170.99 s — — — 4.538, 4.388, 2.012, Pro⁷-CO 1.844, 7.918 73171.32 s — — — 8.138, 4.356, 2.003, Pro⁴-CO 1.805, 4.570 74 173.10 s — —— 4.225, 2.144,1.840 Pro¹⁰-CO 75 173.80 s — — — 2.19, 1.93, 1.733-Gln-δ-CO 173.81 76 173.88 s — — — 7.26, 2.19,1.93,1.73 4-Gln-δ-CO173.86 OH 9.75 br NOE: 6.598 Terpene-2′-OH NH 8.506 d 4.585 NOE:4.869/4.692 His²-NH 8.496 d 4.590 NH 8.206 d 4.484 NOE 3.072, 2.948,4.588 Gln³-NH NH 8.135 d 4.563 His⁵-NH NH 8.110 d 4.470 NOE :4.570,(4.470), Gln⁶-NH 1.727, (1.927) NH 7.918 d 4.534 NOE :4.388,1.827,Leu⁸-NH 1.688, 1.427 NH₂ 7.260 s (6.807) Gln⁶-NH₂ 6.807 s (7.260) NH₂7.230 s (6.789) Gln³-NH₂ 6.789 s (7.230)

In one embodiment of the invention, the compounds of the formula (I) areobtainable by fermentation of Memnoniella echinata FH 2272, DSM 13195,or of one of its variants or mutants under suitable conditions in aculture medium until at least one memno peptide of the formula (I) ispresent in the culture medium, followed by subsequent isolation of thememno peptides.

The invention therefore furthermore relates to a process for thepreparation of a compound of the formula 1, which comprises fermentingthe microorganism Memnoniella echinata FH 2272, DSM 13195, or one of itsvariants or mutants under suitable conditions in a culture medium untilat least one memno peptide of the formula (I) is present in the culturemedium and then isolating the memno peptide from the culture medium.

In one embodiment, the strain FH 2272, DSM 13195, its mutants and/orvariants are fermented in a nutrient solution (also called culturemedium) comprising at least one source of carbon atoms and at least onesource of nitrogen atoms and optionally comprising customary inorganicsalts until memno peptides are present in the culture medium. The memnopeptides may then be isolated from the culture medium and optionallyseparated into the individual active components and purified. In oneembodiment, memno peptides accumulate in the culture medium, and areseparated into individual components.

In another embodiment, at least one source of nitrogen atoms used forthe culture medium is chosen from amino acids and peptides. The aminoacids and peptides used as sources of nitrogen atoms may be the same asthe group (A)n, as defined above.

The process according to the invention can be employed for fermentationon a laboratory scale (milliliter to liter range) and on industrialscale (cubic meter scale).

A strongly producing colony of Memnoniella echinata FH 2272 was grown.An isolate was deposited in the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Mascheroder Weg 1B, D-38124 Braunschweig,Germany, according to the rules of the Budapest Convention on Dec. 9,1999 under the following number: Memnoniella echinata FH 2272, DSM13195.

Memnoniella echinata FH 2272, DSM 13195, has a brown-green mycelium andis characterized by the conidiophores characteristic of the genusMemnoniella.

Variants and mutants of the strain Memnoniella echinata FH 2272, DSM13195, may also be employed to synthesize at least one compound of thememno peptides according to the invention. Such mutants can be producedin a manner known per se by physical means, for example irradiation,such as with ultraviolet rays or X-rays, or chemical mutagens, such asethyl methanesulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOB) orN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Possible variants includethe closely related fungal species Stachybotrys, such as Stachybotrysatra, Stachybotrys chartarum or Stachybotrys complementi.

Screening for mutants and variants which synthesize at least onecompound of the memno peptides according to the invention may be carriedout according to the following scheme:

Separation of the mycelium after fermentation;

Extraction of the mycelium with an organic solvent;

Extraction of the memno peptides from the culture filtrate using solidphases

Analysis by means of HPLC, DC or by testing the biological activity.

The fermentation conditions described below apply to the fungusMemnoniella echinata FH 2272, the deposited isolate DSM 13195 andmutants and variants thereof.

In one embodiment, in a nutrient solution comprising at least one sourceof carbon atoms, casein peptone as a source of nitrogen atoms, andcustomary inorganic salts, Memnoniella echinata FH 2272, produces memnopeptide A. In one embodiment, Memnoniella echinata FH 2272 is DSM 13195.

Possible sources of carbon atoms for aerobic fermentation includeassimilable carbohydrates and sugar alcohols, such as glucose, lactose,sucrose, starches, dextrins, fructose, molasses, glycerol, galactose,and D-mannitol, and carbohydrate-containing natural products, such asmalt extract. Possible sources of nitrogen atoms include, for example,amino acids; peptides; proteins; degradation products of amino acids,peptides and of proteins, such as casein, peptones, and tryptones; meatextracts; yeast extracts; peanut extracts; ground seeds, for example, ofcorn, wheat, beans, soy, and cotton; seed-containing compositions;distillation residues from alcohol production; meat meals; yeastextracts; ammonium salts, and nitrates. In one embodiment, at least onesource of nitrogen atoms is chosen from synthetically obtained peptidesand biosynthetically obtained peptides. The nutrient solution optionallycomprises at least one inorganic salt. In one embodiment, the inorganicsalt is chosen from chlorides, carbonates, sulfates and phosphates. Thesulfates and phospates may be chosen from sulfates of the alkali metals,phosphates of the alkali metals, sulfates of the alkaline earth metals,and phosphates of the alkaline earth metals, wherein the alkaline earthmetals are chosen from iron, zinc, cobalt and manganese.

The formation of the memno peptides according to the invention mayproceed in a nutrient solution comprising casein peptone. In oneembodiment, the nutrient solution comprises a concentration of caseinpeptone ranging from approximately 0.05 to 5%. Another embodimentcomprises a concentration of casein peptone ranging from 0.1 to 1%.Still another embodiment comprises a concentration of casein peptoneranging from 0.2 to 5%. The nutrient solution may comprise glucose. Oneembodiment comprises a concentration of glucose ranging from 0.5 to 3%.The nutrient solution may also comprise cornsteep. In one embodiment thenutrient solution comprises a concentration of corn steep ranging from0.05 to 1%. Another embodiment comprises a concentration of cornsteepranging from 0.1 to 0.5%. The nutrient solution may comprise a trace ofat least one component chosen from potassium chloride, magnesium sulfateand iron sulfate.

In one embodiment, the nutrient solution comprises casein peptone,glucose, cornsteep, and traces of potassium chloride, magnesium sulfateand iron sulfate. In another embodiment, the nutrient solution comprisesa concentration of casein peptone ranging from approximately 0.05 to 5%,a concentration of glucose ranging from 0.5 to 3%, a concentration ofcorn steep ranging from 0.05 to 1%, and traces of potassium chloride,magnesium sulfate and iron sulfate. The data in percent are in each caserelated to the weight of the entire nutrient solution.

In this nutrient solution, Memnoniella echinata, which can beMemnoniella echinata FH 2272, DSM 13195, forms a mixture of memnopeptides. Depending on the composition of the nutrient solution, thequantitative proportion of one or more of the memno peptides accordingto the invention may vary. Moreover, the synthesis of individual memnopeptides can be controlled by the composition of the media such that amemno peptide may not be produced at all or may be produced in an amountbelow the detection limit of the microorganism.

The culturing of the microorganism may be carried out aerobically, i.e.,for example, submerging with shaking or stirring in shaker flasks orfermenters, optionally with introduction of air or oxygen. It can becarried out in a temperature range from approximately 18 to 37° C., anarrower range from approximately 20 to 32° C., and a still narrowerrange of 25 to 30° C. The pH range should be between 6 and 8, such asbetween 6.5 and 7.5. In general, the microorganism is cultured underthese conditions for a period of 24 to 300 hours, more generally 36 to140 hours.

Advantageously, culturing may be carried out in a number of steps, i.e.at least one preculture may be prepared in a liquid nutrient medium, andmay be then inoculated into the actual production medium, the mainculture, for example, in the volume ratio 1:10. The preculture may beobtained, for example, by inoculating a mycelium into a nutrientsolution and allowing it to grow for approximately 36 to 120 hours, suchas for 48 to 72 hours. The mycelium can be obtained, for example, byallowing the strain to grow for approximately 3 to 40 days, such as 4 to10 days, on a solid or liquid nutrient medium, for example, malt-yeastagar or potato dextrose agar (standard medium for mold fungi, forexample, from Difco).

The course of the fermentation can be monitored by means of the pH ofthe cultures or of the mycelium volume as well as by chromatographicmethods, such as thin-layer chromatography or high-pressure liquidchromatography or testing the biological activity. A compound accordingto the invention may be in both the mycelium and the culture filtrate,but the largest part is usually found in the culture filtrate.

The isolation process described below serves for the purification of thememno peptides according to the invention, for example, of memno peptideA. The isolation or purification of a memno peptide according to theinvention from the culture medium may be carried out according to knownmethods taking into account the chemical, physical and biologicalproperties of the natural substances. For the testing of the memnopeptide concentrations in the culture medium or in the individualisolation steps, thin-layer chromatography, for example on silica gel,using isopropanol/25% strength NH₃ as an eluent or HPLC can be used.Detection in thin-layer chromatographic separation can be carried out,for example, by means of color reagents such as chlorosulfonicacid/glacial acetic acid, the amount of the substance formed expedientlybeing compared with a calibration solution.

For the isolation of a memno peptide according to the invention, themycelium is generally first removed from the culture broth using theusual procedures and the memno peptides are then extracted from the cellmass using an optionally water-miscible organic solvent. The organicsolvent phase contains the natural substances according to theinvention; it may be optionally concentrated in vacuo and the residuemay be further purified as described below. In one embodiment, a memnopeptide is extracted from the culture by adding solvent to the mixtureof fungus and broth.

The culture filtrate may be optionally combined with the concentrate ofthe mycelium extract and extracted with a suitable, water-immiscibleorganic solvent, for example with n-butanol. The organic phasesubsequently removed may be optionally concentrated in vacuo. To defatthe valuable products, the concentrate can be diluted with a nonpolarsolvent in which the compounds according to the invention are not verysoluble, for example, with hexane, petroleum ether or diethyl ether. Inthis process, the memno peptides precipitate, and the lipophilicimpurities remain dissolved and may be removed by customary solid/liquidphase separations.

The precipitate comprising the memno peptides may be dissolved in{fraction (1/30)} of the original volume of water/methanol. Theprecipitate dissolves completely in the course of this and may belyophilized. The lyophilizate, subsequently called crude product, maycomprise 5 to 50% memno peptides and may be employed for furtherisolation.

The further purification of at least one memno peptide according to theinvention may be carried out by chromatography on suitable materials,for example, on molecular sieves, on silica gel, alumina, on ionexchangers or on adsorber resins or on reversed phases (RP). The memnopeptides may be separated with the aid of this chromatography. Thechromatography of the memno peptides may be carried out using bufferedaqueous solutions or mixtures of aqueous and organic solutions.

Mixtures of aqueous and organic solutions are understood as meaning allwater-miscible organic solvents, such as methanol, propanol andacetonitrile, in a concentration of 5 to 80% of solvent, more generally20 to 50% of solvent or alternatively all buffered aqueous solutionswhich are miscible with organic solvents. The buffers to be used may bethe same as indicated above.

Separation of the memno peptides on the basis of their differingpolarity may be carried out with the aid of reversed phasechromatography, for example on MCI® (adsorber resin from Mitsubishi,Japan) or Amberlite XAD® (Toso Haas), or further hydrophobic materials,such as on RP-8 or RP-18 phases. Moreover, the separation can be carriedout with the aid of normal-phase chromatography, for example on silicagel, alumina and the like.

Chromatography of the memno peptides may be carried out using bufferedor acidified aqueous solutions or mixtures of aqueous solutions withalcohols or other, water-miscible organic solvents. In one embodiment,the organic solvent is chosen from propanol and acetonitrile.

Buffered or acidified aqueous solutions are understood as meaning atleast one solution alone or in combination. For example, said at leastone solution may be chosen from water, phosphate buffers, ammoniumacetate, citrate buffers, and acids. In one embodiment, citrate bufferis in a concentration ranging from 0 to 0.5 M. Acids are chosen fromformic acid, acetic acid, trifluoroacetic acid, and all commerciallyavailable acids known to the person skilled in the art. In oneembodiment, the commercially available acid is in a concentrationranging from 0 to 1%. In yet another embodiment the concentration ofacid is 0.1%.

Chromatography may be carried out using a gradient which begins with100% of water and ends with 100% of solvent. At least one solvent isused. A mixture of two or more solvents may also be used. In oneembodiment, a linear gradient is run from 20 to 50% of a solvent chosenfrom propanol and acetonitrile.

Alternatively, gel chromatography or chromatography on hydrophobicphases can also be carried out.

Gel chromatography may be carried out on polyacrylamide or copolymergels, such as Biogel-P 2® (Biorad) or Fractogel TSK HW 40® (Merck,Germany or Toso Haas, USA).

A further, very effective process for the purification of the compoundsaccording to the invention may be the use of ion exchangers. Forexample, the basic memno peptide A can be isolated very advantageouslyon cation exchangers, such as Fractogel® EMD SO₃. Buffer solutionsbetween pH 5 and 8, such as between pH 6 and 7.5, can be used. Elutioncan be achieved, for example, using a rising salt gradient. In additionto water, it can also be advantageous to use mixtures of aqueous buffersolutions with an organic solvent as solvents. The proportion of theorganic solvent may be between 10% and 90%, such as between 30 and 60%.

The sequence of the abovementioned chromatographic processes may bereversible.

A further, very effective purification step for memno peptides iscrystallization. Memno peptides may be crystallized out from solutionsin organic solvents and from mixtures of water with organic solvents.Crystallization may be carried out in a manner known per se, for exampleby concentrating or cooling saturated memno peptide solutions.

The memno peptides according to the invention are stable in the solidstate and in solutions in the pH range between 3 and 8, for example 5and 7, and can thus be incorporated into customary pharmaceuticalpreparations.

The formation of the nitrogen-containing memno peptides can be favoredby adding the desired amino acids or peptides as precursor to theMemnoniella echinata cultures. It may be the peculiarity of theMemnoniella echinata species that they bind the amino groups of theamino acids and peptides supplied with synthesis of a five-membered ringlactam, usually of an isoindole ring system. This binding takes placeeither starting from the aldehyde precursor in the course of anoxidation or from the lactone oxidation stage in ring-opened form or thecyclic lactone form. This ability of the Memnoniella echinata to bindamines is completely surprising and can be used for obtaining protected,converted amine derivatives, such as of terpene derivatives of aminoacids and peptides.

At least one compound of the memno peptides according to the inventionmay be suitable on account of their valuable pharmacological propertiesfor use in human or veterinary medicine as pharmaceuticals.

The present invention thus relates to the use of the compound of theformula (I) or a physiologically tolerable salt thereof for theproduction of a cardiac remedy for the treatment or prophylaxis ofcardiac insufficiency.

The compounds according to the present invention may also be used forthe production of a pharmaceutical for the treatment of diseases whereincardiac insufficiency is a primary or secondary cause of the diseasesuch as, e.g., circulatory insufficiency.

The mechanism of action of the memno peptides is uncertain, but asignificant effect was detected.

For the detection of the activators of SERCA2 which imitate thephosphorylation effect of PLB, a calorimetric test may be run whichdetermines the activity of the Ca²⁺ ATPase in dog heart microsomes inthe presence of test substances. The test may be carried out in 96-wellmicrotiter plates. The enzymatic release of inorganic phosphate fromATP, which forms a blue colored complex with ammonium molybdate whichcan be measured at 620 nm in a spectrophotometer, may be measured.

Memno peptide A activates SERCA2 in concentrations from 12.5 μM.

The memno peptides moreover have an antimicrobial action.

The present invention thus relates to the use of the compound of theformula (I) or a physiologically tolerable salt thereof for theproduction of a pharmaceutical for the treatment of microbial, forexample bacterial, infections.

Table 3 shows some minimum inhibitory concentrations (MIC) of theantimicrobial spectrum of memno peptide A against some selectedbacteria.

TABLE 3 Resistance MIC Strain against: [μg/ml] Staphylococcus aureus SG511 — 16 Staphylococcus aureus 285 Penicillin 16 Staphylococcus aureus503 Penicillin 16 Staphylococcus aureus FH 1982 Methicillin 16Staphylococcus aureus 701E Methicillin 16 Staphylococcus aureus 707EMethicillin 16 Staphylococcus aureus 9 Tüb Ofloxacin 16 Staph.epidermidis ZH 2c — 16 Staph. epidermidis 763 Methicillin >16 Staph.epidermidis 5747IIW Methicillin 32 Staph. epidermidis 291 Ofloxacin 8Staph. epidermidis 799 Ofloxacin 8 Enterococcus faecium Md8B — 8Enterococcus faecium VR1 Vancomycin >64 Enterococcus faecium VR2Vancomycin >64 Streptococcus pyogenes VR3 Vancomycin >64 Streptococcuspyogenes 308A — 8 Streptococcus pyogenes 77A — 4

In addition to the antibacterial action, the compounds according to theinvention exhibit weak antimycotic, i.e. antifungal, properties, forexample against Candida albicans.

Moreover, the substances according to the invention have a certainfavorable inhibitory action on glucose-6-phosphate translocase(G-6-P—TL), an enzyme which is of importance for glucose metabolism andthus for the treatment of diabetes mellitus. For example, the compoundmemno peptide A exhibits selective activities against G-6-P—TL, but itdoes not inhibit the coenzyme G-6-P phosphatase.

The present invention thus relates to the use of the compound of theformula (I) or a physiologically tolerable salt thereof for theproduction of a pharmaceutical for the therapy of diabetes mellitus.

The invention also relates to pharmaceutical preparations of at leastone compound of the memno peptides according to the invention.

At least one compound of the memno peptides according to the invention,can generally be administered as such in undiluted form. Use as amixture with suitable excipients or vehicles is one embodiment of theinvention. Vehicles which can be used in pharmaceuticals may be thecustomary and pharmacologically tolerable vehicles and/or excipients.

In general, the pharmaceuticals according to the invention may beadministered orally or parenterally, but rectal administration is alsopossible. Suitable solid or liquid pharmaceutical preparation forms are,for example, granules, powders, tablets, coated tablets,(micro)capsules, suppositories, syrups, emulsions, suspensions,aerosols, drops or injectable solutions in ampoule form and preparationshaving protected release of active compound, in whose preparationvehicles and additives and/or excipients such as disintegrants, binders,coating agents, swelling agents, glidants or lubricants, flavorings,sweeteners or solubilizers are customarily used. Frequently usedvehicles or excipients which may be mentioned are, for example,magnesium carbonate, titanium dioxide, lactose, mannitol and othersugars, talc, lactoprotein, gelatin, starch, vitamins, cellulose and itsderivatives, animal or vegetable oils, polyethylene glycols andsolvents, such as sterile water, alcohols, glycerol and polyhydricalcohols.

If appropriate, the dose units can be microencapsulated for oraladministration in order to delay release or to extend it over a longerperiod of time, for example by coating or embedding the active compoundin particle form in suitable polymers, waxes or the like.

In one embodiment, the pharmaceutical preparations may be produced andadministered in dose units, each unit comprising, as active constituent,a specific dose of at least one compound of the memno peptides accordingto the invention. In the case of solid dose units such as tablets,capsules and suppositories, this dose can be up to approximately 500 mg,but more generally can be approximately 0.1 to 200 mg, and in the caseof injection solutions in ampoule form up to approximately 200 mg, butusually approximately 0.5 to 100 mg, per day.

The daily dose to be administered is generally dependent on the bodyweight, age, sex and condition of the mammal. Under certaincircumstances, however, higher or lower daily doses may also beappropriate. Administration of the daily dose can be carried out both bysingle administration in the form of an individual dose unit or else ina number of smaller dose units and by multiple administration ofsubdivided doses at specific intervals.

The pharmaceuticals according to the invention may be produced bybringing at least one compound of the memno peptides according to theinvention into a suitable administration form using customary vehiclesand, if appropriate, additives and/or excipients.

The invention is illustrated further in the following examples.Percentages relate to weight. Mixing ratios in the case of liquids referto the volume, if no other details have been given.

The following examples are intended to illustrate the invention withoutlimiting the scope thereof.

EXAMPLES Example 1

Preparation of a Glycerol Culture of Memnoniella echinata FH 2272, DSM13195.

100 ml of nutrient solution (malt extract 2.0%, yeast extract 0.2%,glucose 1.0%, (NH₄)₂ HPO₄ 0.05%, pH 6.0) in a sterile 300 ml Erlenmeyerflaskwere inoculated with the strain Memnoniella echinata FH 2272, DSM13195, and incubated on a rotating shaker at 25° C. and 140 rpm for 7days. 1.5 ml of this culture were then diluted with 2.5 ml of 80%strength glycerol and stored at −20° C.

Example 2

Preparation in an Erlenmeyer Flask of a Culture or a Preculture ofMemnoniella echinata FH 2272, DSM 13195.

A sterile 300 ml Erlenmeyer flask containing 100 ml of the followingnutrient solution, 10 g/l of glucose, 5 g/l of casein peptone, 1.7 g/lof liquid cornsteep, and 7 ml of trace element solution (10 g/l of KCl,10 g/l of MgSO₄×7 H₂O, 3.6 g/l of FeSO₄×7 H₂O and 6 g/l of MgSO₄×H₂O)was inoculated with a culture grown in a slant tube (same nutrientsolution, but with 2% agar) or with 1 ml of a glycerol culture (seeExample 1) and incubated at 180 rpm and 30° C. on a shaker. The maximumproduction of at least one compounds of the memno peptides according tothe invention was achieved after about 120 hours. For the inoculation of10 and 200 l fermenters, a submerged culture 48 to 96 hours old(inoculation quantity about 10%) from the same nutrient solutionsufficed.

Example 3

Preparation of the Memno Peptides.

A 30 l fermenter was operated under the following conditions:

Nutrient medium:  10 g/l of glucose 0.5 g/l of casein peptone 1.7 g/l ofliquid cornsteep 7 ml of trace element solution pH 6.5 (beforesterilization) Trace element solution: KCl 10 g/l, MgSO₄ × 7 H₂O 10 g/l,FeSO₄ × 7 H₂O 3.6 g/l and MnSO₄ × H₂O 6 g/l Incubation time: 45 hoursIncubation temperature: 28° C. Stirrer speed: 300 rpm Aeration: 15 lmin⁻¹

Foam formation was optionally suppressed by repeated addition ofethanolic polyol solution. The production maximum was achieved afterabout 35 to 70 hours.

Example 4

Isolation of the Memno Peptide Mixture from the Culture Solution ofMemnoniella echinata FH 2272, DSM 13195.

After completion of the fermentation of Memnoniella echinata FH 2272,DSM 13195, the culture broth of the fermenter, obtained according toExample 3 (200 liters) was filtered with addition of about 2% filter aid(e.g. Celite®) and the cell mass (22 liters) was extracted with 66liters of methanol. The methanolic solution containing the valuablesubstance was freed of the mycelium by filtration and concentrated invacuo. The concentrate was diluted with water and applied to a prepared,17 liter MCI GEL, CHP20P column together with the culture filtrate (180liters). It was eluted with a gradient of water after 60% propan-2-ol inwater. The column flow (25 liters per hour) was collected in fractions(10 liters each) and the memno peptide-containing fractions (from 25% to30% propan-2-ol) were combined.

Concentration in vacuo afforded 20 liters of a brown solution. Sixliters of cation exchanger, Fractogel® EMD SO₃, equilibrated at pH 7with potassium phosphate buffer were packed into a column (125 mm×500mm). After loading the ion exchanger with 20 liters of the concentratedescribed above, it was eluted with a gradient of 10 mM potassiumphosphate buffer, pH 7, after 1 M NaCl in 10 mM potassium phosphatebuffer, pH 7 in water/methanol (1:1). The column flow, i.e. the unboundmaterial, contained the neutral memno peptides (49 g). The column flowwas 12 liters per hour; 1 liter portions were collected in fractionsduring the gradient elution. With 0.75 M NaCl (fractions 31 and 32),memno peptide A was obtained. Fractions 31 and 32 were combined andconcentrated to approximately 500 ml in vacuo.

Example 5

Enrichment of Memno Peptide A by Gel Chromatography.

8 g of the product obtained according to Example 4 were applied to acolumn of 3.9 liters capacity packed with Fractogel® TSK HW-40 s(width×height=10 cm×50 cm). The eluent:methanol/water (1:1) was pumpedthrough the column at a flow rate of 20 ml per minute and the columnoutflow was collected in fractions (20 ml). The memno peptides A werefound mainly in fractions 75 to 85. They were combined and freed of themethanol in vacuo. They afforded 0.9 g of active compound mixture.

Example 6

HPLC System for the Detection of the Memno Peptides.

The system described below allowed purity testing and also separationand quantification of the memnoterpenes, for example in the crudemixture or in the culture filtrates.

Eluent: 0.1% trifluoroacetic acid in 32% acetonitrile.

Column: Nucleosil 100C₁₈AB 250/4, Macherey-Nagel.

Flow: 1.0 ml/min

Detection: Ultraviolet light absorption at 210 nm.

Under the conditions indicated, memno peptide A can thus have thefollowing retention time:

Memno peptide A: 7.0 minutes.

Example 7

Purification of Memno Peptide A.

500 ml of solution of memno peptide A isolated and enriched according toExample 5 were applied to a 500 ml Nucleosil®100-7 C₁₈AB column andchromatographed using a gradient of 25 to 50% acetonitrile in 0.05%trifluoroacetic acid/water. The flow of the eluent was 50 ml per minute;the fraction size 50 ml. Memno peptide A was found in fractions 71 to88. Repeated purification of the combined fractions with a constantsolvent concentration of 28% of acetonitrile in 0.05% trifluoroaceticacid afforded>95% of pure memno peptide C after freeze drying (100 mg).

Characterization of Memno Peptide A:

10 μg of memno peptide A were hydrolyzed in constant-boilinghydrochloric acid and investigated in an amino acid analyzer. Thefollowing customary amino acids were found:

Glutamic acid 11 nMol Proline 22 nMol Histidine 10 nMol Leucine 5.6 nMolMethionine oxide 5.5 nMol

Ultraviolet absorption: λ_(max) at 269 nm, 305 nm (shoulder).

The high-resolution FAB mass spectrum showed an intensive MH⁺ at m/z1565.7819 Da, in good agreement with the calculated mass (forC₇₆H₁₀₉N₁₆O₁₈S, monoisotopic) of 1565.7827 Da. The MS/MS fragmentationcorresponded to the formula (IVa).

1 1 10 PRT artificial sequence misc_feature Description of ArtificialSequence Memnoniella echinata, FH 2271, DSM 1319 1 Met His Gln Pro HisGln Pro Leu Pro Pro 1 5 10

What is claimed is:
 1. A compound of formula V

wherein R₁ and R₂ together are double bonded O, or are each H, or H andOH, or H and O—C₁-C₄-alkyl: R₃ and R₄ together are double bonded O, orare each H, or H and OH, or H an O—C₁-C₄-alkyl: R₅ and R₇ are each H: R₈is chosen from H, OH, C₁-C₄-alkyl, and O—C₁-C₄-alkyl: R₁₀ is H orglycosidically bonded sugar; A is a natural amino acid selected fromGly, Ala, Val, Leu, lle, Pro, Ser, Thr, Phe, Tyr, Trp, Lys, Arg, Asp,His, Glu, Asn, Gln, Cys, and Met: n is an integer chosen from 1 to 12,wherein each A in A(n) is the same or different from every other A:wherein the nitrogen atom in the isoindole ring of formula V is theN-terminal amine nitrogen of the first acid of the A(n) group; or a saltthereof.
 2. A compound according to claim 1, or a salt thereof, wherein(A)n is a peptide chain having 2 to 12 natural amino acids as defined inclaim
 1. 3. A compound according to claim 1, or a salt thereof ,wherein(A)n is a peptide chain having the amino acid sequence:Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro (SEQ ID NO: 1) wherein the Metis optionally oxidized to form a sulfoxide.
 4. A compound according toclaim 1, or a salt thereof, wherein R₁₀ is an aldohexose.
 5. A compoundaccording to claim 1, or a salt thereof, obtainable by cultivation ofMemnoniella echinata FH 22727, or DSM 13195, under suitable conditionsin a culture medium until a compound according to claim 1 is present inthe culture medium, and said compound is subsequently isolated.
 6. Acompound according to claim 5, further comprising conversion of saidcompound into at least one physiologically tolerable salt.
 7. Acompound, memno peptide A, C₇₆H₁₀₈N₁₆O₁₈S, of the formula IVa:

or a salt thereof.
 8. A compound according to claim 1, wherein thegroup-(A)n comprises a part of the amino acid sequence for casein.
 9. Acompound according to claim 8, wherein (A)n is the amino acid sequence:Met His Gln Pro His Gln Pro Leu Pro Pro (SEQ ID NO:1).
 10. A compositioncomprising at least one compound of formula V, wherein said compound isdefined according to claim 1, and optionally a vehicle, excipient, orcombinations thereof.
 11. A composition according to claim 10, whereinsaid composition is a pharmaceutical composition and said vehicle,excipient, or combinations thereof are pharmaceutically acceptable. 12.A composition comprising at least one compound of formula IVa, asdefined in claim 7, and optionally a vehicle, excipient, or combinationsthereof.
 13. A composition according to claim 12, wherein saidcomposition is a pharmaceutical composition and said vehicle, excipient,or combinations thereof are pharmaceutically acceptable.
 14. A processfor making a compound of formula V, or a salt thereof, as defined inclaim 1, comprising cultivation of Memnoniella echinata FH 2272, or DSM13195, under suitable conditions in a nutrient solution comprising atleast one source of carbon atoms and at least one source of nitrogenatoms until at least one compound of formula V is present in thenutrient solution and said compound is subsequently isolated.
 15. Aprocess according to claim 14, further comprising, conversion of thecompound into at least one physiologically tolerable salt.
 16. A processaccording to claim 14, wherein said cultivation occurs under aerobicconditions.
 17. A process according to claim 14, wherein said at leastone source of nitrogen atoms is chosen from amino acids and peptides.18. A process according to claim 14, wherein said nutrient solutioncomprises casein peptone at a concentration ranging from about 0.05% to5% by weight of the nutrient solution.
 19. A process according to claim14, wherein said nutrient solution comprises glucose at a concentrationranging from 0.5% to 3% by weight of the nutrient solution.
 20. Aprocess according to claim 14, wherein said nutrient solution comprisescorn steep at a concentration ranging from 0.05 to 1% by weight of thenutrient solution.
 21. A process according to claim 14, wherein saidnutrient solution comprises casein peptone, glucose, cornsteep, andtraces of potassium chloride, magnesium sulfate and iron sulfate.
 22. Aprocess according to claim 14, wherein said cultivation is carried outat a temperature ranging from about 18 to 37° C.
 23. A process accordingto claim 14, wherein said cultivation is carried out at a pH rangingfrom 3.0 to 8.0.
 24. A process according of claim 14, wherein saidcultivation is carried out during a time period ranging from 24 to 300hours.
 25. A process according to claim 14, wherein said cultivation iscarried out in submerged conditions.
 26. A process according to claim14, wherein said compound is isolated from at least one component chosenfrom culture filtrates and mycelia.
 27. A process for treating cardiacinsufficiency in a mammal, comprising administering to the mammal inneed thereof an effective amount of at least one compound of formula V,or a salt thereof, as defined in claim
 1. 28. A process for treatingcardiac insufficiency in a mammal, comprising administering to themammal in need thereof an effective amount of at least one compound offormula IVa, or a salt thereof, as defined in claim
 7. 29. A process fortreating a disease in a mammal wherein cardiac insufficiency is aprimary or secondary cause, comprising administering to the mammal inneed thereof an effective amount of at least one compound of formula V,or a salt thereof, as defined in claim
 1. 30. A process for treating adisease in a mammal wherein cardiac insufficiency is a primary orsecondary cause, comprising administering to the mammal in need thereofan effective amount of at least one compound of formula IVa, or a saltthereof, as defined in claim
 7. 31. A process for treating diabetesmellitus in a mammal, comprising administering to the mammal in needthereof an effective amount of at least one compound of formula V, or asalt thereof, as defined in claim
 1. 32. A process for treating diabetesmellitus in a mammal, comprising administering to the mammal in needthereof an effective amount of at least one compound of formula IVa, ora salt thereof, as defined in claim
 7. 33. A process for treating amicrobial infection in a mammal, comprising administering to the mammalin need thereof an effective amount of at least one compound of formulaV, or a salt thereof, as defined in claim
 1. 34. A process for treatinga microbial infection in a mammal, comprising administering to themammal in need thereof an effective amount of at least one compound offormula IVa, or a salt thereof, as defined in claim 7.